Cystic fibrosis: typing 48 German families with linked DNA probes

Summary Two hundred and thirty five subjects from 48 German cystic fibrosis (CF) families were typed for restriction fragment length polymorphisms (RFLPs) detected by the probes pmet H, pmet D, and pJ 3.11, known to be tightly linked to the CF gene. Gene and haplotype frequencies suggest a linkage disequilibrium with the CF locus. The analysis of the predictive value of this typing in individual CF families indicates that the combined use of these probes provides a powerful diagnostic system both for carrier detection and prenatal diagnosis. In 33 out of 48 families carriers and non-carriers could be identified, and in 26 of these 33 families prenatal diagnosis could discriminate between affected and unaffected offspring.     

Cystic fibrosis: typing 89 German families with linked DNA probes

Summary Three hundred and ninety-two subjects from 89 German families were typed for restriction fragment length polymorphisms (RFLPs) detected by the probes pmetH, pmetD, pJ3.11, KM19, and XV2c known to be tightly linked to the cystic fibrosis (CF) gene. The analysis of the predictive value of this typing in individual CF families indicates that the combined use of these probes provides a powerful diagnostic system for both carrier detection and prenatal diagnosis. In 45 families the complete haplotype including all RFLPs was available. Of them 41 (91.1%) were fully informative and 4 were partly informative.     

Cystic fibrosis: screening for a DNA deletion by field inversion gel electrophoresis

Summary We analyzed DNA of ten cystic fibrosis (CF) patients representing DNA of 19 different CF chromosomes and screened for deletions by means of field inversion gel electrophoresis (FIGE). No differences were detected after digestion of the DNA samples with the restriction enzymes Not I and Sfi I and hybridization with the probes MetH, MetD, J3.11, and 7C22. Thus the percentage of deletions occurring within the CF region and detectable with FIGE is less than 15.2% (95% confidence interval, N=19)     

Cystic fibrosis in Greece: Typing with DNA probes and identification of the common molecular defect

Summary The relative frequency of the ΔF508 mutation in the Greek population is 54.1%; this is similar to that reported in other Southern European populations and contrasts with the considerably higher frequencies encountered in Northern Europe and North America. The low frequency is in agreement with the linkage disequilibrium already reported between cystic fibrosis and haplotype B in this country. In contrast to the common association of pancreatic insufficiency with the homozygous ΔF508 genotype, the present study revealed two homozygous children with no evidence of pancreatic failure.     

Cystic fibrosis newborn screening: a pilot study to maximize carrier screening

Newborn screening for cystic fibrosis (CF) is expanding because early diagnosis has been shown to result in improved nutrition and growth. Most newborns identified by a mutation panel have a single detected mutation and require sweat testing to exclude an additional undetected mutation. The resulting identification of CF carrier newborns, although not the primary purpose of screening, has three potential benefits, (1) the detection of trait-trait couples, (2) presymptomatic testing of these couples' previously born children who may have undetected CF, and (3) a carrier parent alerting his/her extended family members to the chance of also being a CF carrier. Reaping each benefit requires genetic counseling of parents and their accepting carrier testing. The purpose of this study was to utilize the sweat testing visit to educate parents about the value of carrier testing for themselves and their blood relatives. We compared special care (genetic counseling after explaining the sweat test result and offering of parental DNA testing, all on the sweat test visit) versus standard care (sweat test result reported by phone to the parents the next day by the newborn's physician, ideally with the recommendation to arrange genetic counseling and parental carrier testing). In the first year of New York State CF screening, 64 newborns with one detected mutation were reported in the nine-county region that includes Rochester. Of these, parents of 39 agreed to participate in the study and to be randomized to special or standard care. Sixty-one parents completed both the initial and 1-year follow-up questionnaires (30 couples and one mother). Of the 61 parents, 23 had carrier testing after the birth of the baby. The frequency of such parental testing was significantly higher in the special care group (17/34 or 50%) than in the standard care group (6/27 or 22%) (p < 0.05). This is the first evidence from a randomized trial that genetic counseling and offering carrier testing to parents on the sweat test visit increases identification of carrier parents. Such identification detects trait-trait parents and facilitates carrier testing among relatives.     

Cystic fibrosis: a disorder with defective autophagy

The accumulation of misfolded and/or ubiquitinated protein aggregates with a perturbation of autophagy has been described in several human pathologies. A sequestration of misfolded cystic: fibrosis transmembrane conductance regulator (CFTR) and cross-linked PPARγ has been observed in airway epithelia of cystic fibrosis (CF) patients. CF airways are also characterized by chronic inflammation, pro-oxidative environment and increased transglutaminase 2 (TG2) levels. We showed that defective CFTR drives autophagy inhibition through reactive oxygen species (ROS)-TG2- mediated aggresome sequestration of the Beclin 1 interactome. Rescuing Beclin 1 at the level of the endoplasmic reticulum and autophagy favors clearance of aggresomes, improves CFTR trafficking and ameliorates CF lung inflammation both in vitro and in vivo. Therefore, rescuing autophagy interrupts the vicious cycle linking defective CFTR and lung inflammation and may pave the way to the development of a novel class of drugs for the treatment of CF.     

Cystic fibrosis testing in a referral laboratory: results and lessons from a six-year period

Background

The recent introduction of high throughput sequencing technologies into clinical genetics has made it practical to simultaneously sequence many genes. In contrast, previous technologies limited sequencing based tests to only a handful of genes. While the ability to more accurately diagnose inherited diseases is a great benefit it introduces specific challenges. Interpretation of missense mutations continues to be challenging and the number of variants of uncertain significance continues to grow.

Results

We leveraged the data available at ARUP Laboratories, a major reference laboratory, for the CFTR gene to explore specific challenges related to variant interpretation, including a focus on understanding ethnic-specific variants and an evaluation of existing databases for clinical interpretation of variants. In this study we analyzed 555 patients representing eight different ethnic groups. We observed 184 different variants, most of which were ethnic group specific. Eighty-five percent of these variants were present in the Cystic Fibrosis Mutation Database, whereas the Human Mutation Database and dbSNP/1000 Genomes had far fewer of the observed variants. Finally, 21 of the variants were novel and we report these variants and their clinical classifications.

Conclusions

Based on our analyses of data from six years of CFTR testing at ARUP Laboratories a more comprehensive, clinical grade database is needed for the accurate interpretation of observed variants. Furthermore, there is a particular need for more and better information regarding variants from individuals of non-Caucasian ethnicity.     

Identification and epidemiological typing of Naegleria fowleri with DNA probes.

Naegleria fowleri is a small free-living amoeboflagellate found in warm water habitats worldwide. The organism is pathogenic to humans, causing fatal primary amoebic meningoencephalitis. When monitoring the environment for the presence of N. fowleri, it is important to reliably differentiate the organism from other closely related but nonpathogenic species. To this end, we have developed species-specific DNA probes for use in the rapid identification of N. fowleri from the environment. Samples were taken from the thermal springs in Bath, England, and cultured for amoebae. Of 84 isolates of thermophilic Naegleria spp., 10 were identified as N. fowleri by probe hybridization. The identity of these isolates was subsequently confirmed by their specific whole-cell DNA restriction fragment length polymorphisms (RFLPs). One DNA clone was found to contain a repeated element that detected chromosomal RFLPs that were not directly visible on agarose gels. This enabled the further differentiation of strains within geographically defined whole-cell DNA RFLP groups. N. fowleri DNA probes represent a specific and potentially rapid method for the identification of the organism soon after primary isolation from the environment.     

Cystic fibrosis DNA markers in Alabama blacks

DNA samples from unrelated Alabama blacks with no family history of cystic fibrosis (CF) were analyzed with DNA markers linked to cf. Allelic frequencies of genetic markers detected by probes pmetH, pmetD, XV2c, KM19, and pJ3.11 were compared to those of other populations. Allelic frequencies for pJ3.11, XV2c, and KM19 in Alabama blacks and previously studied Caucasian populations were similar. In contrast, the met locus allelic frequencies in Alabama blacks were markedly different from those in Caucasian populations.     

Cystic fibrosis heterozygote screening in 5,161 pregnant women.

A screening program for cystic fibrosis (CF) heterozygotes was conducted in a large HMO prenatal population, to evaluate the level of interest among eligible patients, the effectiveness of prescreening education, attitudes toward the screening process, psychological effects, and utilization of prenatal diagnosis and its outcomes. The heterozygote identification rate and frequency of specific CFTR mutations were also assessed. Identified carriers were offered genetic counseling and testing of male partners. Prenatal diagnosis was offered if both parents were identified as carriers. A total of 5,161 women underwent carrier testing; 947 others completed survey instruments only. The acceptance rate of screening was high (78%), and pretest education by videotape was generally effective. Adverse psychological effects were not reported. Participants generally found screening to be desirable and useful. Screening identified 142 female heterozygotes, 109 couples in which the male partner was not a carrier, and 7 high-risk couples. The incidence of R117H mutations was much higher than expected. The number of identified carriers was much lower in Hispanics than in Caucasians. We conclude that large-scale prenatal screening for CF heterozygotes in the absence of a family history of CF is an acceptable method for identifying couples at risk for affected fetuses. Sufficient pretest education can be accomplished efficiently, test insensitivity is well accepted, adverse psychological events are not observed, and general patient satisfaction is high.     

Cystic fibrosis: a mucosal immunodeficiency syndrome

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a channel that regulates the transport of ions and the movement of water across the epithelial barrier. Mutations in CFTR, which form the basis for the clinical manifestations of cystic fibrosis, affect the epithelial innate immune function in the lung, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pulmonary pathogens. Compounding the effects of excessive neutrophil recruitment, the mutant CFTR channel does not transport antioxidants to counteract neutrophil-associated oxidative stress. Whereas mutant CFTR expression in leukocytes outside of the lung does not markedly impair their function, the expected regulation of inflammation in the airways is clearly deficient in cystic fibrosis. The resulting bacterial infections, which are caused by organisms that have substantial genetic and metabolic flexibility, can resist multiple classes of antibiotics and evade phagocytic clearance. The development of animal models that approximate the human pulmonary phenotypes-airway inflammation and spontaneous infection-may provide the much-needed tools to establish how CFTR regulates mucosal immunity and to test directly the effect of pharmacologic potentiation and correction of mutant CFTR function on bacterial clearance.     

Cystic fibrosis: A look into the future of prenatal screening and therapy

Despite recent guidelines suggesting prenatal screening for carriers of cystic fibrosis (CF) mutations, many physicians do not offer patients this service or even counseling. Some argue that the risks of miscarriage associated with prenatal diagnostic techniques outweigh the benefit of added insight, but with the advent of newer, noninvasive techniques, risks of miscarriage may be significantly lowered. Prenatal diagnosis provides parents the time to prepare for raising a child with CF, and soon, could provide treatment options in utero that could improve quality of life. Here, we describe two of the most promising gene therapy approaches: lentivirus and adenoassociated virus (AAV)‐mediated gene transduction. Thus, prenatal detection and treatment is in a most crucial stage for care of patients with CF. Birth Defects Research (Part C) 105:73–80, 2015. © 2015 Wiley Periodicals, Inc.     

Cystic fibrosis: a disease in electrolyte transport

Cystic fibrosis (CF) is a fatal genetic disease caused by abnormalities in fluid and electrolyte transport in exocrine epithelia. Both absorptive and secretory processes are affected by an underlying membrane defect in Cl- permeability. However, the impact of the defect on transport function is tissue specific. Net electrolyte absorption is decreased in the sweat duct, increased in airway epithelia, and not affected in intestine. The defect is expressed in secretion as a consistent failure in most, if not all, exocrine tissues, to beta-adrenergically stimulated and cAMP mediated secretory response. However, the secretory response to cholinergic and Ca2(+)-mediated stimulation is normal in the sweat gland, apparently normal in the airway, but absent in the intestine. The basic defect is not fatal in and of itself, and the imbalance between absorption and secretory functions may be of some selective advantage to heterozygotes in surviving complications of intestinal infections. The inherent defect in transport is probably the primary physiological cause of the ultimately fatal secondary infections in the lungs of CF homozygotes.     

Rapid typing of HLA-DQB1 DNA polymorphism using nonradioactive oligonucleotide probes and amplified DNA

The allelic sequence diversity at theHLA-DQBI locus has been analyzed by polymerase chain reaction (PCR) amplification and sequencing. Fifteen amino acid sequence-defined alleles (one previously unreported) and several silent nucleotide polymorphisms which subdivide these alleles have been identified. Here, we describe the specific amplification of theDQB1 second exon by several different PCR primer pairs and a simple and rapid typing procedure using a panel of 16 horseradish peroxidase (HRP)-labeled oligonucleotide probes capable of distinguishing theseDQBI alleles.